Abstract

Objectives: Low socioeconomic status (SES) is one of the most robust social factors associated with disease morbidity, including more
severe asthma in childhood. However, our understanding of the biological processes that explain this link is limited. This
study tested whether the social environment could get “under the skin” to alter genomic activity in children with asthma.

Design and participants: Two group design of children with physician diagnosed asthma who came from low or high SES families.

Outcomes: Genome-wide transcriptional profiles from T lymphocytes of children with asthma.

Results: Children with asthma from a low SES background showed overexpression of genes regulating inflammatory processes, including
those involved in chemokine activity, stress responses and wound responses, compared with children with asthma from a high
SES background. Bioinformatic analysis suggested that decreased activity of cyclic AMP response element binding protein and
nuclear factor Y and increased nuclear factor κB transcriptional signalling mediated these effects. These pathways are known
to regulate catecholamine and inflammatory signalling in immune cells.

Conclusions: This study provides the first evidence in a sample of paediatric patients diagnosed with asthma that the larger social environment
can affect processes at the genomic level. Specifically, gene transcription control pathways that regulate inflammation and
catecholamine signalling were found to vary by SES in children with asthma. Because these pathways are the primary targets
of many asthma medications, these findings suggest that the larger social environment may alter molecular mechanisms that
have implications for the efficacy of asthma therapeutics.

The larger social environment has been found to affect physical health in individuals across the lifespan. Across all social
factors, low socioeconomic status (SES) exhibits one of the strongest and most consistent associations with morbidity and
mortality across a wide range of diseases,1 including childhood asthma. In particular, children with asthma from a low SES background suffer from more frequent hospitalisations,
emergency department visits and more functional impairment (eg, activity limitations, days spent in bed) compared with children
from a higher SES background.2–8

In seeking to understand how the larger social environment “gets in the body” to impact on the pathophysiology of asthma,
researchers have investigated relationships between the social environment and inflammation. In healthy adults, low SES has
been linked to heightened levels of circulating inflammatory markers, such as C reactive protein and interleukin (IL)6.910 In children with asthma, low SES has been associated with greater eosinophil counts as well as heightened in vitro stimulated
production of inflammatory cytokines implicated in asthma, such as IL5 and IL13.1112 Another social factor associated with low SES—stress—has been linked to heightened inflammatory responses to allergen challenge
in patients with asthma,13 and decreased expression of genes coding hormonal receptors that regulate inflammation, including the glucocorticoid and
β adrenergic receptors.14

Although relationships between SES and inflammation are increasingly recognised, the molecular mechanisms underlying this
relationship remain unclear. The present study utilised an in vivo genomics based approach to identify differences in T cell
gene expression associated with SES in a sample of children with asthma, and to identify candidate signal transduction pathways
that may shape those differences. Because the focus was on understanding disparities associated with disease progression,
we targeted a sample of children with pre-existing asthma.

Based on previous research linking SES and inflammation, we hypothesised that low SES would be associated with alterations
in proinflammatory transcription control pathways. Based on previous data implicating the β adrenergic and glucorticoid pathways
in the relationship between social factors and inflammation,14 we hypothesised that transcriptional mediators of these types of hormonal signals would differentiate T cell gene expression
profiles in children from low versus high SES backgrounds. As a second aim, we tested whether social factors related to SES,
such as cognitive interpretations, could explain associations between SES and genome-wide transcriptional profiles. Our hypothesis
was that SES colours the way in which children interpret their social world, and that these interpretations in turn alter
neuroendocrine and inflammatory signalling processes indicated by genome-wide transcriptional profiles.

METHODS

Additional details are available in the supplement (online only).

Participants

Participants were recruited from an ongoing longitudinal study of children with asthma aged 9–18 years. A subset of 16 children
from low SES and 15 from high SES backgrounds provided blood samples for this study. The low and high SES groups were identified
based on those who fell into the bottom and top 15% on parent education and family income variables.

Measures

Background characteristics

Family SES was measured by parent report of annual family income and years of parental education. Medical information included
use of asthma medications and asthma severity.

Interpretations of stress

The Cognitive Appraisal and Understanding of Social Events (CAUSE) videos depict age appropriate ambiguous life situations,
such as being asked to talk with a teacher after class.15 Children’s interpretations are rated by judges, with higher numbers indicating greater perceived threat.

Transcriptional mediation analyses

We used a two sample variant of the Transcription Element Listening System (TELiS) (http://www.telis.ucla.edu)17 to analyse differential gene expression data in terms of the prevalence of transcription factor binding motifs (TFBMs) within
the promoters of differentially expressed genes, as previously described.20

Ancillary analyses used a multivariate generalisation of the analysis of covariance (ANCOVA)21 to control for potential confounders which could explain associations between SES and gene expression profiles. Covariates
included demographic variables (age, gender), medical variables (asthma severity, use of inhaled corticosteroid medication,
use of β agonist medication) and interpretations of stress (CAUSE video responses). To confirm that natural killer cell prevalence
did not affect the results, the relative concentration of mRNAs encoding CD16 and CD56 were included as covariates.

RESULTS

Descriptive information about the sample is shown in table 1. The low and high SES groups did not differ in terms of asthma severity, use of inhaled corticosteroids, use of β agonists,
forced expiratory volume in 1 s or atopic status (all p values >0.15).

Differential gene expression

Sixty transcripts were differentially expressed between the low and high SES groups, representing 56 distinct named human
genes. Thirty-four reflected overexpression in the low SES group whereas 26 reflected overexpression in the high SES group
(table 2).

A second finding involved decreased prevalence of NF-Y TFBMs in promoters of genes overexpressed in children from a low SES
background (average 72% decrease in promoter TFBM prevalence, p = 0.022). NF-Y is activated by the same cAMP/PKA signalling
pathway as CREB, providing a convergent indication of pathway activation.

Decreased activity of AP1 (Fos/Jun) transcription factors in T cells from children from a low SES background was also found
(AP1 p = 0.046; VJUN p = 0.048). Reduced activity of this pathway is consistent with the observed downregulation of the FOS, FOSB and JUN transcripts, which encode key elements of the AP1 transcription factor family.

All of the above findings remained significant in sensitivity analyses parametrically varying promoter length and TFBM detection
stringency (table 4). Other TFBMs were identified in initial analyses as potential contributors to the observed SES related differences in gene
expression. However, these TFBMs did not show consistent statistical significance across parametric sensitivity analyses (p
values >0.05) (table 4). To assess the likelihood that the observed differences might arise by chance, we drew 10 000 random samples of 60 transcripts
from the population of genes assayed by the Affymetrix U133A high density oligonucleotide array, and computed the magnitude
of differential prevalence for the 10 TFBMs identified in initial analyses. Results confirmed that the observed differential
distributions of CREB, NF-Y, AP1 and NFκB were unlikely to occur in a random sample of genes (p<0.05) (table 4).

Confounders

After controlling for age and gender, patterns of associations with CREB, NF-Y and NFκB remained statistically significant
(all p values <0.045), with one exception which was the relationship between SES and CREB controlling for age (p = 0.068).
Associations also remained significant after controlling for asthma severity, inhaled corticosteroid use and β agonist use
(all p values ⩽.05). Unlike the other transcription control pathways, indications of association between SES and AP1 activity
were rendered non-significant by control for several confounders, including age (p = 0.52), gender (p = 0.15) and β agonist
use (p = 0.11).

Controlling for cognitive interpretations reduced the relationship between SES and CREB activity to non-significant (p = 0.25).
The same was true for the relationship between SES and NFκB (p = 0.12) and to a lesser extent SES and NF-Y (p = 0.077). These
analyses suggest that children from the low SES group were more likely to perceive threat in ambiguous situations, and this
tendency may activate neuroendocrine processes (impacting on CREB and NF-Y through the cAMP/PKA pathway) that ultimately impact
on inflammatory signalling pathways (eg, NFκB).

Finally, controlling for the prevalence of mRNAs encoding CD16 and CD56 did not significantly alter the results for CREB,
NF-Y or NFκB (all p values <0.05), suggesting that results are not simply a function of different relative proportions of
T cell versus NK cell subtypes in each group.

DISCUSSION

The present study provides the first empirical evidence linking the larger social environment (SES) to T cell gene expression
in the context of a clinical inflammatory disease. Among children with asthma, those from a low SES background showed overexpression
of genes regulating a variety of inflammatory processes, including chemokine activity, stress responses and wound responses.
Promoter based bioinformatics analyses identified transcription control pathways that may structure the observed patterns
of differential gene expression, including decreased CREB, AP1 and NF-Y, and increased NFκB signalling. Children in the low
SES group also reported poorer clinical outcomes, such as greater asthma symptoms. Although the cross sectional nature of
this study precludes definitive conclusions about causal direction, the findings are consistent with the hypothesis that the
larger social environment can get “under the skin” at the level of genomic transcription control pathways.

Genes overexpressed in children in the low SES group included: chemokine ligands such as CXCL4 and CXCL7 which recruit and
activate leucocytes;23 those involved in antigen processing and presentation (MHC class II protein complexes), a key feature in asthma inflammatory
biology; those related to oxidative stress (SOD2), which recruits and activates immune cells, prolonging inflammation; and those related to calcium binding proteins (S100A8, S100A9, S100A12), which have chemotactic effects on leucocytes and proinflammatory effects on endothelial cells.2425 These patterns suggest molecular regulatory mechanisms that may heighten inflammation and worsen clinical outcomes in children
with asthma from a low SES background.

Genes overexpressed in children in the high SES group included: those encoding heat shock proteins (HSPA1A, HSPA1B, HSPA6, DNAJB1), which protect cells from inflammatory molecules such as reactive oxygen species, and are protective against pulmonary inflammation2627; those involved in cell differentiation and proliferation, such as c-fos and c-jun, which are increased in response to exposure
to reactive oxygen species28; those related to cell cycle control (BTG2), which helps contain the effects of DNA damage29; and those involved in the methylation, or silencing, of genes. Note that FOS, FOSB and JUN gene products also encode key components of the AP1 family of transcription factors, which in promoter based bioinformatic
analyses showed increased activity in the high SES group. Taken together, these patterns suggest that among children with
asthma from a high SES background, heightened inflammation may be counterbalanced by cellular regulatory mechanisms aimed
at containing the damage due to inflammation.

This study also identified candidate transcription control pathways that may orchestrate differential patterns of gene expression
as a function of SES. Bioinformatic analyses indicated downregulated activity of CREB, AP1 and NF-Y, and upregulated NFκB
mediated transcription in children with asthma from a low SES background. CREB mediates transcriptional responses to β adrenergic
receptor signalling through the adenylyl cyclase/cAMP/protein kinase A pathway.2230 Adenylyl cyclase activity is impaired after allergen challenge in patients with asthma.31 Diminished regulatory signalling from cAMP pathways could lead to increased activation of T cells, and subsequent expression
of Th-2 cytokines.31 Reduced CREB signalling may also decrease the efficacy of bronchodilators used as therapeutic agents in asthma. These patterns
are consistent with decreased β adrenergic receptor mRNA found in lymphocytes of children with asthma with chronic and acute
life stress.14

Bioinformatic analyses also indicated reduced NF-Y mediated transcription in children with asthma from a low SES background.
Like CREB, NF-Y is phosphorylated by PKA, and may thus serve as an indicator of signalling along the β adrenergic signalling
pathway.32 The fact that both transcription factors showed downregulation in the low SES group suggests multiple parallel deficiencies
across catecholamine signalling pathways.

Bioinformatic analyses also indicated upregulated NFκB signalling in children with asthma in the low SES group. NFκB transactivates
a wide variety of inflammatory mediators. Some data suggest that elevated cAMP activity can inhibit NFκB activity33; hence increased NFκB and decreased CREB signalling could represent a common regulatory alteration that shifts gene expression
profiles towards a more inflammatory phenotype in children from low SES backgrounds.

Our findings are consistent with previous research that has investigated functional immune measures. For example, children
with asthma from a low SES background show greater production of Th-2 cytokines and greater eosinophil counts compared with
those from a high SES background.12 Greater stress has been linked to in vivo inflammatory responses to allergen challenge and in vitro cytokine production in
patients with asthma1334 and predisposed to allergic disease.35 Furthermore, stress has been linked to reduced expression of genes coding for hormonal receptors that regulate inflammation.14

Statistical analyses revealed that interpretations of stress accounts for some of the SES related differences in indicators
of CREB and NFκB activity. This suggests that in order for the social environment to have biological effects, it may have
to first be perceived in a threatening manner. In turn, these cognitive perceptions may come with biological costs in transcriptional
regulation and inflammatory biology. We note there could be numerous other pathways linking SES to genomic patterns, which
should be tested in future studies.

It is unclear whether similar effects would be found in healthy children. However, even if similar effects were evident, these
biological mechanisms could still have different implications for those with a pre-existing inflammatory disease. Nonetheless,
it would be important for future research to test the effects of SES on gene expression profiles in healthy individuals as
well.

Limitations include the observational design, precluding conclusions about causality and directionality. This is a necessary
limitation to human work involving social factors that are difficult to randomly assign (eg, socioeconomic status). Secondly,
the sample size is small by epidemiological standards, although not unusual for genome-wide microarray studies. One concern
about small samples is insufficient power; however, the significant differences in several transcription control pathways
suggest that the magnitude of effects is large enough to be detected in a sample of this size. A second concern is whether
the findings will be robust and reliable. However, note that the bioinformatics approach used in this study capitalises on
the data from thousands of genes to form aggregate and more reliable indicators of transcription factor activity. Thirdly,
microarray technology is sometimes criticised as being too exploratory. In this study, however, microarray data were used
to test an a priori hypothesis of SES increases in inflammation due to specific hormonal signalling pathways documented in
previous research.1214 Furthermore, the use of false discovery rate analysis is a standard approach in the genomic literature for adjusting statistics
to account for multiple comparisons. Nevertheless, replicating these results in other samples would be important in future
studies. Finally, selection of the CD2 antigen to isolate T lymphocytes may have permitted contamination by natural killer
(NK) cells. (CD3 markers were not used because the immunomagnetic separation process for CD3 would have activated T cells.)
However, the present results were not significantly altered in analyses controlling for NK cell marker mRNA.

The present data identified a distinct transcriptional fingerprint of low SES environments, and candidate transcription control
pathways structuring those differences, in T lymphocytes from children with asthma. Children with asthma from a low SES background
showed overexpression of genes related to inflammation, chemokine activity, and stress and wound responses, and bioinformatics
indications of reduced CREB, AP1 and NF-Y, and increased NFκB signalling. This study provides the first clinical evidence
in asthma that broader social environments affect processes at the genomic level, specifically in terms of transcription control
pathways that regulate inflammation and catecholamine signalling. Because these pathways are the primary targets of many asthma
medications, these findings suggest that the larger social environment may also affect the efficacy of asthma therapeutics.
Finally, perceptions of stress play an important role in explaining how SES gets transduced into alterations at the genomic
level. Overall, these findings provide new insights into the mechanisms by which social factors affect the course of inflammatory
diseases, and highlight the need for future research investigating how the pathophysiology of asthma is shaped by social environments.